Stable radioiodine conjugates and methods for their synthesis

ABSTRACT

Methods are described for conjugating radioiodinated peptides to non-metabolizable carbohydrates with improved yields and qualities of conjugates. Radioiodinated residualizing antibody conjugates comprising a carbohydrate-appended peptide are also provided. The instant radioiodinated residualizing antibody conjugates are particularly stable in vivo and are suitable for radioimmunodetection and radioimmunotherapy.

This application is a continuation in part of U.S. Ser. No. 08/919,477filed on Aug. 28, 1997 now U.S. Pat. No. 6,558,669, which claims benefitof No. 60/024,738, filed Aug. 28, 1996.

FIELD OF THE INVENTION

This invention relates to the preparation of reagents used inradioimmunodetection and radioimmunotherapy and specifically to thepreparation of radioiodine labeled conjugates having enhanced stabilityin vivo and enhanced retention at tumor sites.

BACKGROUND OF THE INVENTION

Radioiodinated monoclonal antibodies are important for the diagnosis andtherapy of cancer as summarized by Goldenberg in Amer. J. Med. 94:297-312 (1993). A number of methods have been developed over the lastthirty years to chemically introduce radioiodine into monoclonal andpolyclonal antibodies for these uses. Iodine is preferred as aradiolabel in these applications because the chemistry used forradioiodination of protein is relatively easy, radioiodine has usefulphysical decay characteristics, and isotopes of iodine are commerciallyavailable.

Among useful iodine isotopes are Iodine-124, which has been used toradiolabel antibodies as described by Pentlow et al., Journal of NuclearMedicine, 37: 1557-62 (1996), and Iodine-125, which has been used fordetection using an intraoperative probe as described by Martin et al.,Cancer Investigation, 14: 560-71 (1996). In the context of using theseiodine isotopes, one concern is the long circulation time ofradioiodinated antibodies, which leads to high background radiation. Thehigh background problem is compounded by the loss of radioiodine fromtarget cells, when standard radioiodination methods are used. A poortarget to non-target ratio of delivered iodine often results from thehigh background and radioiodine loss problems. Accordingly, a principalaim in the art is to improve the target to non-target ratio. Iodine-125has been proposed for therapy purposes because of its cascade Augerelectrons as described by Aronsson et al., Nuclear Medicine and biology,20: 133-44 (1993). Clearly, optimum use of a long-lived [t_(½), 60 days]low energy-emitting nuclide demands that intracellular target retentionbe achieved; which is not possible with conventional radioiodinationmethods.

Various chemistries have been developed to link iodine to antibodiesthat target cancer cells. These chemistries have been reviewed byWilbur, Bioconjugate Chemistry 3: 433-70 (1992). The most common linkingprocedure has been to prepare in situ an electrophilic radioiodinespecies to react with a functional group on an antibody. Reagents suchas chloramine T and iodogen have been employed to generate electrophiliciodine. A tyrosine group on protein is usually the site of iodination.However, the presence of a harsh oxidant or reductant may lead tostructural impairment of an antibody. For this reason, an alternativeapproach is to iodinate a small organic molecule and couple the pureiodinated species to antibody. N-Succinimidyl3-(3-iodo-4-hydroxyphenyl)propionate (Bolton-Hunter reagent) is anexample of the latter category. These and other methods have beenreviewed by Wilbur (Id).

A major drawback with using the foregoing radioiodination schemes is thephenomenon of in vivo deiodination. As a result of antibodyinternalization and lysosomal processing in vivo, a labelled protein isdegraded to small peptides, and its radioiodine is released from thecell in the form of iodotyrosine or as iodine attached to a lowmolecular weight peptide fragment. These findings have been reported byGeissler et al., Cancer Research 52: 2907-2915 (1992) and Axworthy etal., J. Nucl. Med. 30: 793 (1989). Such in vivo removal of radioiodinefrom target cells has a profound bearing on the use of iodine isotopesfor radiodiagnosis and radiotherapy. Discrimination between tumor andnon-tumor that is relevant to diagnosis and therapy, and the prolongedretention of isotope on a tumor cell, relevant to radiotherapy, areseverely compromised by the occurrence of in vivo deiodination. This isreadily appreciated if one considers the 8-day half-life of iodine-131,which is widely used for radioimmunotherapy investigations. Ifantibodies radioiodinated with this isotope are metabolized withconsequent removal of the isotope from the target cells within the first24-120 h post-injection of the reagent, the advantage of the lengthyhalf-life of this isotope for therapy is lost. That is, the usefulhalf-life of this isotope is not exploited in a prolonged tumoricidaleffect because of the above-described drawback of conventionalradioiodination chemistry.

In contrast to this drawback of conventional chemistry, the action of invivo deiodinases in releasing iodine in the form of molecular iodinefrom the cell is less significant to the problem of optimizing thetarget to non-target ratio of radioisotope accumulation. Workers in thisfield have attempted to prepare iodinated proteins that do not‘deiodinate’ by the action of in vivo deiodinases as reviewed by Wilburet al., Journal of Nuclear Medicine, 30: 216-26 (1989). But theseattempts have failed to show improvement in cellular retention ofradioiodine. The reason for these failures is that, contrary to what wasexpected, the metabolic clearance of intact iodotyrosine was moreimportant to clearance of isotope than was the deiodination of tyrosineto liberate radioiodine.

One way to overcome the unacceptably fast release of radioiodine fromconjugate is to attach iodine to non-metabolizable carbohydrate and toconjugate the resultant entity to antibodies. After antibody catabolismwithin a tumor cell, the radioiodine remains stably attached to thecarbohydrate and thus is trapped inside the cell. These carbohydratelabels, referred to as ‘residualizing labels’, are exemplified byStrobel et al., Arch. Biochem. Biophys 240: 635-45 (1985) and Ali etal., Cancer Research (suppl) 50: 783s-88s (1990). However, thesemethods, when applied to the labelling of monoclonal antibodies (Mabs),suffer from one or both of the following drawbacks: (1) Very lowradiolabeling yields (3-6%) and (2) formation of aggregates (up to 20%).Low conjugation yield necessitates handling a large amount ofradioactive iodine to incorporate sufficient radioactive label inantibody. This approach causes a radiation safety concern as well aswastage of most of the unusable radioactivity. As a result, the specificactivity achieved by this method suffers. Furthermore, aggregateformation can lead to reduced tumor uptake and will lead to enhancedliver uptake, and thereby impair the effectiveness of the radiolabelmethod. The full advantage of using residualizing labels forradioimmunodetection and radioimmunotherapy cannot be realized unlessprogress can be made to limit the twin problems of poor radiolabelingyield and aggregate formation when using carbohydrate-based reagents.Using novel substrates and methodologies to address these issues isanother aspect of this invention.

One well known approach to this problem is to label the antibody with aradiometal ion such as indium-111 or an isotope of yttrium, using abifunctional aminopolycarboxylate ligand such as bifunctional EDTA orbifunctional DTPA. These radiolabelled conjugates exhibit prolongedretention of radiometal in tumor as exemplified in in vivo animalexperiments by Stein et al., Cancer Research 55: 3132-39 (1995). Thatis, radiometal ions chelated to aminopolycarboxylates also behave, invivo, as residualizing labels. Thus, the problem of residualizationgenerally applies to techniques that use these labels as well.

The prior art has addressed the issue of residualizing iodine labels byusing non-metabolizable sugars to which an iodinatable group isattached. An iodinatable group such as tyramine is reductively coupledto the carbohydrate, so that there is no metabolizable peptide bondbetween tyramine and the sugar entity. There are two main problemsencountered with these prior art methods. These are in theantibody-coupling steps. One method, that of Strobel et al. (see above),uses a carbohydrate-adduct derived from lactose, and couples proteinsand antibodies to the same by first oxidizing the galactose portion ofsuch adducts with galactose oxidase. Usually poor overall yield (3-6%)is obtained, as described by Stein et al. Cancer Research, 55:3132-3139, (1995). Furthermore, lactose is an inefficient substrate forgalactose oxidase. In examining a number of galactose-containingcarbohydrate derivatives for their ability to be oxidized by thisenzyme, Avigad et al. (J. Biol. Chem 237: 2736-2743, (1962)), determinedthat lactose had less than half the affinity of D-galactose forgalactose oxidase, and was oxidized fifty times slower compared togalactose. This inefficient step therefore contributes to overallreduced radioisotope incorporation into antibodies.

Another approach involved in coupling to antibodies does not make use ofany special property such as the ability of the carbohydrate to beselectively derivatized by an enzyme (such as galactose oxidaseoxidation involving galactose moiety), but makes use of cyanuricchloride as the cross-linker to link both the iodinated carbohydrate andantibody. This approach has the serious problem of generating antibodyaggregates. Cyanuric chloride has been used to form conjugates butunfortunately this reagent contains three reactive chlorines andconsequently forms aggregates. Another factor involved in aggregateformation is the presence of multiple amino residues in antibodies thatcan bind to the residualizing agent and/or coupling reagent,particularly with carbohydrate residualizing agents that couple toprotein by reductive amination. Such multiple binding causes aggregatesto form, and results in low specific activity of radiolabel in theprepared conjugate mixture. Accordingly, coupling agents are needed thatdo not cause aggregate formation.

SUMMARY OF THE INVENTION

The present invention solves the above-identified problems by providingpreparation methods and compositions of iodinatable peptides consistingof unnatural D-amino acid components. The radioiodinated versions ofthese conjugates are used to label antibodies and produce residualizinglabels.

The present invention also is directed to the design of bifunctionaliodinatable aminopolycarboxylate adducts wherein the iodinatable groupis attached to the said aminopolycarboxylate unit via anon-metabolizable peptide bond. The adducts are radioiodinated andconjugated to Mabs or their fragments, and thereby introduceresidualizing label into biospecific Mabs.

The present invention additionally is directed toward the design of newmethodologies to improve yields in the radioiodination of monoclonalantibodies using carbohydrate-based reagents.

The present invention is further directed to the design ofcarbohydrate-based reagents which improve the quality of residualizinglabel-antibody conjugates (with minimal aggregation) and therebydecrease non-target accumulation (especially in liver) of the label invivo.

The present invention is also directed toward the ready attachment ofsuch residualizing radioiodine labels to targeting vectors, includingproteins such as monoclonal antibodies, fragments and constructsthereof.

In one embodiment, nonmetabolizable and radioiodinated peptides are usedfor labeling antibodies so that the radioactivity is residualized invivo. These specially designed hydrophilic peptides preferably have amolecular weight of more than 500 (i.e. 5 amino acid residues or more).More preferably, the peptide has a molecular weight of between 1000 and4000 (10 to 40 amino acid residues) although in some cases more than 40amino acids are acceptable. A hydrophilic peptide in the context usedhere means that the peptide contains polar amino acid units that arecharged, such as aspartic acid, glutamic, acid, lysine and arginine orthat are polar, such as serine and threonine. The presence of multiplehydrophilic acid groups from these residues and their nonmetabolizablepeptide bonds allow residualization of the radiolabel after antibodycatabolism by lysosomes. Most preferred in this context are acidic aminoacid residues such as aspartic acid.

D-amino acids comprise the peptide between the site of attachment of thepeptide to an antibody and a radioactive iodine that is bound to atyrosine or tyramine. Most particularly, within this region, no twoadjacent amino acids are L-amino acids. Glycine in this context is anL-amino acid. By using D-amino acids in this way, the peptide bonds thatconnect the radioactive iodine to the antibody cannot be hydrolyzed in alysosome.

In a second embodiment, a bifunctional aminopolycarboxylate systemcontaining an iodinatable group is prepared by first synthesizing apeptide unit consisting of two differentially protected amino groups andunnatural D-amino acid units in the peptide mer. Sequential elaborationof the amino groups by adding an aminopolycarboxylate unit and thenadding a protein cross linker completes the synthesis of thebifunctional aminocarboxylate. The peptide contains one or moreunnatural D-tyrosine units. The amino acid units of the peptide areattached via non-metabolizable amide bonds. The antibody-binding groupcan be an amino residue (for site-specific attachments to oxidizedcarbohydrate of MAbs), an imidate or isothiocyanate (attachable tolysine groups of proteins), maleimide, bromo- or iodoacetamide residue(specific to thiols on Mabs) and the like. The number of amino acidunits in the peptide is two to ten, preferably three, of which at leastone is D-tyrosine. The amino acid(s) immediately following the lastD-tyrosine unit, and which are used to introduce antibody-bindingcross-linkers, can be natural L-amino acids. The aminopolycarboxylateunit can be iminodiacetic acid, nitrilotriacetic acid, EDTA(ethylenediaminetetraacetic acid), DTPA (diethylenetriaminetetraaceticacid), TTHA (triethylenetetraminehexaacetic acid), DOTA(1,4,7,10-tetraaza cyclododecane N,N′,N″N″′-tetraacetic acid) or variousbackbone-substituted versions thereof, such as, for example,isothiocyanatobenzyl-EDTA/DTPA/TTHA/DOTA, among numerous otheraminopolycarboxylates and their derivatives which can be readilyenvisaged.

In a third embodiment, the bifunctional iodinatable aminopolycarboxylateis derived by attaching a tyramine group and an antibody-binding groupto the aminopolycarboxylate. No protease-susceptible bond is involved inthese structures. Alternatively, aminopoly-carboxylates,backbone-substituted with an antibody-binding unit, are converted tocorresponding dianhydrides which are then reacted with D-tyrosine toobtain an entity that contains two D-tyrosine residues. Since the amidebond(s) between the bifunctional aminopolycarboxylate and D-tyrosinewill not be recognized by proteases, these constitute a differentversion of residualizing iodine labels.

One key feature in all of these systems is that the iodinated D-tyrosinemoiety will be resistant toward deiodinases. This possibility isdescribed by Dumas et al., Biochem. Biophys. Acta 293: 36-47 (1973).

In a fourth embodiment, a carbohydrate-based residualizing label isdesigned using a disaccharide which contains a galactose unit and whichcan be oxidized readily with galactose oxidase. This embodiment isexemplified by a preparation derived from melibiose. Another example isprovided wherein the carbohydrate-based residualizing label is preparedwhich already contains an antibody-binding group. Yet another aspect inthis regard involves using hydrazide-appended antibodies for reactionwith iodinated and derivatized carbohydrate.

In a fifth embodiment, a radioiodinated carbohydrate is allowed to reactwith a cyanuric chloride derivative which in turn is already derivatizedto possess antibody-binding moiety.

A sixth embodiment involves conjugating a reducing sugar to a peptidecomprising one or more D-tyrosine units. The resulting product isfurther derivatized to incorporate a protein binding group andradioiodine. In particular, this embodiment is exemplified bycarbohydrates appended to peptides and is useful for radioiodinating anantibody, comprising: (a) a peptide that comprises at least oneD-tyrosine, an amino terminus, a carboxy terminus formed from a D-lysineand no contiguous L-amino acids between the D-tyrosine and the carboxyterminus; (b) a carbohydrate conjugated to the peptide via an E-aminogroup of the D-lysine to form a carbyhydrate-appended peptide; and (c) alinker group for covalently binding said carbohydrate-appended peptideto an antibody.

Methods of the invention provide greater efficiencies of antibodylabeling with residualizing iodine labels. The methods also providehigher quality stable radioiodine conjugate preparations having a lowaggregate content. Other objects and advantages will become apparentfrom the following detailed description.

DETAILED DESCRIPTION

The present invention solves the problems of poor labeling efficiencyand aggregate formation reported in the carbohydrate-based prior art intwo general ways. In the first way, a new method is provided that allowsoxidation of the galactose-containing carbohydrate-tyramine (orD-tyrosine) adduct by galactose oxidase. The invention achieves this byusing melibiose as the carbohydrate in the adduct. The affinity ofmelibiose for galactose oxidase is five times as high as that ofgalactose and ten-times as high compared to the affinity of lactose forgalactose oxidase. Furthermore, melibiose is oxidized at a ratecomparable to galactose. Consequently, this method of the inventionenhances the overall process yield obtained in the oxidation step.Overall incorporations of 18.7-20.7% (see Example-9) have been achievedfor the radioiodination of antibody using radioiodinated and oxidized(oxidation using galactose oxidase) dimelibiitoltyramine of the presentinvention. These incorporations are five-to-ten fold higher than yieldsobserved in the radioiodination of the same antibody, usingradioiodinated and oxidized dilactitoltyramine. An advantage of thepresent invention in this regard lies in utilizing a substrate(dimelibiitoltyramine) which is oxidized readily by galactose oxidase.An additional invention in this context involves the use ofhydrazide-appended antibodies which results in enhanced yield in thestep of reductive coupling of carbohydrate addend to proteins.

The second way that the invention solves the prior art problemsmentioned above is to improve the quality of iodinated antibodyconjugate prepared by cyanuric chloride-mediated protein-carbohydratecoupling. This is achieved two ways: (1) by introducing one, or alimited number of more reactive hydrazide residues into an antibody thatreacts preferentially with the coupling reagent, instead of the morenumerous protein primary amine residues; and (2) by using cyanuricdichloride derivatives to couple antibody to residualizing label. Asused in the invention, monosubstituted cyanuric chloride, prepared undernon-aqueous conditions, carries a thiol-reactive entity such asmaleimide, and is used for coupling to thiolated antibody. The secondchlorine of this cyanuric dichloride is used to react with a phenolichydroxyl group, such as that from a tyramine residue, while the thiolgroup of thiolated antibody reacts with maleimide group in a subsequentstep. The third chlorine is unreactive, and is not a factor. Aggregateformation is therefore minimized and specific activity of the preparedconjugates is improved.

According to one aspect of the invention a radioisotope of iodine isattached in a non-metabolizable manner to a substrate. The iodine thenbecomes trapped within the acidic environment of lysosomes afterantibody catabolism. The consequent prolonged retention of radioiodinewithin a target cell such as a tumor cell facilitates target organdosimetry and enhanced target to non-target discrimination. In thecontext of tumor targeting this enhancement allows more effectiveradiodiagnosis and radiotherapy. Another aspect of the invention is anew class of peptide-based residualizing labels. These residualizinglabels address problems encountered in the use of carbohydrate-basediodine labels.

The use of peptide-based residualizing labels involves peptidesconsisting of one or more unnatural D-tyrosine units that are bonded toother unnatural amino acids. These peptides preferably containhydrophilic amino acids such as D-aspartic acid and D-glutamic acidunits for increased hydrophilicity, and are of at least 5 amino acidresidues in size. The amino terminal residue of these peptides can be anL- or D-amino acid, provided that if an L-amino acid, it is not directlyattached to a tyrosine, and can be attached to a protein-binding crosslinker for later attachment to an antibody. Once radioiodinated andcoupled to antibody, the iodinated peptide unit is residualized within acell lysosome after attachment to a cell surface via binding andprocessing of the associated antibody. The presence of non-metabolizableamide bonds, hydrophilic amino acid residues such as charged asparticacid residues and glutamic acid residues, and a size greater than 4amino acid residues collectively enable such residualization. Mostpreferred in this context is the use of aspartic acid residues for thepeptide.

The conjugate's structure can be varied by using D-lysine at the peptidecarboxyl terminus. This provides an ε-amine group for attaching anaminopolycarboxylate such as nitrilotriacetic acid (NTA),ethylenediamnine-tetraacetic acid (EDTA), diethylenetriaminepentaaceticacid (DTPA) or triethylenetetraminehexaacetic acid (TTHA). In thisembodiment, the ε-amine at the carboxyl end and the amine at the amineterminus are differentially protected for attaching anamino-polycarboxylate and a cross-linker at designated loci. In thisvariation of the invention, D-aspartic acids in the peptide areoptionally substituted with other amino acids. Yet another variation isthe rational design of aminopolycarboxylate systems which contain aradioiodinatable group such as tyramine as well as an antibody-bindingmoiety.

Products of the present invention deal with structural aspects whichconfer enhanced stability after the antibody is internalized andprocessed. It is this antibody processing that leads to diminishedretention of radioiodine in tumor, which is exacerbated withinternalizing antibodies. Our invention addresses this issue by thedesign of a non-metabolizable peptide template which is also attached toaminopolycarboxylate and an iodinatable entity. By attaching polargroups such as DTPA to D-lysine which in turn is attached to D-tyrosinewhich is coupled to a protein binding moiety, the invention ensures thatthe entire piece of aminocarboxylate-D-lysine-[I-125]-D-tyrosine portionwill be trapped in lysosomes, after antibody processing, by virtue ofthe presence of protease-resistant peptide bonds, hydrophilic nature andthe size. This is in contrast to iodotyrosine, the catabolite ofconventionally radioiodinated antibody, which readily escapes from thelysosomes and causes reduced radioactivity retention at the tumor sites.

Another related class of stable radioiodine agents involves the productof reductive amination of a carbohydrate to the side chain of an aminogroup of a basic D-amino acid, which in turn is bound to a polypeptidecomprising one or more D-tyrosine units, followed by furtherderivatization of this product to incorporate a linker group for bindingto an antibody. The carbohydrate may be a non-metabolizable reducingsugar, disaccharide or oligosaccharide. Preferably, the carbohydrate isa non-metabolizable reducing sugar, such as melibiose or lactose.

The resulting non-metabolizable carbohydrate-appended peptideconstitutes an improvement over traditionally used dilactoltyramine(‘DLT’)-type structures. First, the overall efficiency of Mab labelingis enhanced by an order of magnitude, up to 50%, at a specific activityof greater than 2 mCi/mg, with less than 2% aggregation. With thesesignificant improvements in overall efficiency in yield and quality oflabeling Mabs, the instant methods are available for practicalapplications. Second, the process is simplified in that the entirelabeling is carried out as a one-vial procedure. There is no need foractivating the radioiodinated substrates for conjugation to Mabs. Third,the method is flexible to accommodate structural variations, since thepeptide backbone can be readily modified for regulating in vivopharmacokinetics of Mabs labeled in this manner. Lysosomalresidualization of Mabs radioiodinated by the methods of the inventionis due to the stable attachment of radioiodinated D-tyrosine to a sugarnonmetabolizable in humans.

In this connection, the instant invention also provides an agent usefulfor radioiodinating an antibody, comprising: (a) a peptide thatcomprises at least one D-tyrosine, an amino terminus, a carboxy terminusformed from a D-lysine, and no contiguous L-amino acids between theD-tyrosine and the carboxy terminus; (b) a carbohydrate conjugated tothe peptide via an ε-amino group of the D-lysine to form acarbohydrate-appended peptide; and (c) a linker group for covalentlybinding said carbohydrate-appended peptide to an antibody.

The invention therefore also includes antibody conjugates comprising acarbohydrate-appended peptide of the instant invention covalently boundto an antibody through a linker, and radioiodinated conjugates.

DEFINITIONS

In the description that follows, a number of terms are utilizedextensively. Definitions are provided here to facilitate understandingof the invention.

Phosphate Buffer

As used herein, “phosphate buffer” refers to an aqueous solution of 0.1M sodium phosphate that is adjusted to a Ph between 6.0 and 7.5.

Antibody

As used herein, “antibody” includes monoclonal antibodies, such asmurine, chimeric, humanized or human antibodies, as well asantigen-binding fragments thereof. Such fragments include Fab, Fab′,F(ab)₂, and F(ab′)₂, which lack the Fc fragment of an intact antibody.Such fragments also include isolated fragments consisting of the lightchain variable region, “Fv” fragments consisting of the variable regionsof the heavy and light chains, (sFv′)2 fragments (see, for example: Taiet al., Cancer Research Supplement, 55:5983-5989, 1995), and recombinantsingle chain polypeptide molecules in which light and heavy variableregions are connected by a peptide linker.

Radioiodine-antibody Conjugate

As used herein, a radioiodine-antibody conjugate is a moleculecomprising at least one residualizing label and an antibody. Aradioiodine-antibody conjugate retains the immunoreactivity of theantibody, i.e., the antibody moiety has roughly the same, or onlyslightly reduced, ability to bind antigen after conjugation compared tobinding before conjugation with the residualizing label.

Residualizing Label

“Residualizing label” is a radiolabel that is covalently attached toprotein and has been designed to remain entrapped within lysosomes oranother subcellular compartment following degradation of the carrierprotein. Generally, residualizing labels are synthesized from moleculeswhich themselves are not readily degraded in lysosomes. In general,these tracers have been radioactive carbohydrates or metal chelates ofaminopolycarboxylates such as DTPA.

Any non-metabolizable carbohydrate is suitable for the presentinvention. Dimelibiitoltyramine (DMT) and melibiitoltyramine (MT) aresome examples in this category. Bifunctional DTPA (or EDTA), eitheralone or as a D-tyrosine appended substrate, exemplifies the category ofaminopolycarboxylates.

Aggregate

As used herein, an “aggregate” is a molecular complex comprising atleast one extra polypeptide in addition to the desired antibody. Theextra polypeptide is coupled directly or indirectly to the antibody bycovalent means. Examples of aggregates are dimers, trimers and othermultimers of the antibody. Macromolecular complexes comprised of morethan one residualizing label per antibody can be considered aggregatesif by virtue of excessive labeling of antibody by residualizing label,the antibody binding activity is compromised. But the labeling of anantibody by multiple residualizing labels is often desired as a means toincrease the specific radioactivity of the prepared antibody conjugate.

Five embodiments of the present invention are shown in SCHEMES I-VIIbelow and are described seriatim.

SCHEME I

ABG-[CL]-AA-[(D)-AA]_(m)-[(D)Tyr]_(n)-(D)-Lys-OH

Where m and n are each integers and m+n=4-40, AA represents an aminoacid, D denotes a D-amino acid, CL is a cross linker and ABG is anantibody-binding group. The design of a peptide containing one or moreD-tyrosine residues, one or more hydrophilic amino acids such asD-aspartic acid and other amino acids is achieved by using a resin. TheFmoc protected first amino acid (optionally shown as D-lysine) isanchored via its carboxyl end to a resin support such as from achlorotrityl-chloride resin. The peptide is elaborated by sequentialaddition of amino acids, each amine is protected by a Fmoc group and thecarboxylic acid is activated. After forming each amide bond, the Fmocgroup is removed. This removal allows coupling to the nextcarboxyl-activated Fmoc-protected D-amino acid. The assembly of peptidesis thus a straightforward procedure. After liberating the final peptidefrom the solid support, the amine terminus is attached to a suitableheterobifunctional or homobifunctional cross-linker. Many of thesecross-linkers are commercially available. One or more hydrophilic aminoacids such as aspartic acid are introduced to increase thehydrophilicity of the peptide. The peptide thus formed has a minimumsize of 5 amino acids. A metabolically stable D-tyrosine-containinghydrophilic peptide made in accordance with the invention is useful as aresidualizing iodine label.

A variant of the above theme is to prepare a peptide that contains aD-lysine at its carboxyl terminus and attach the ε-amino group of thislysine to an aminopolycarboxylate such as, for example, EDTA, DTPA, andthe like. In the general structure shown in SCHEME II, m is an integerhaving a value of 0, 1 or 2. In the peptide portion, the letter Ddenotes D amino acid, AA stands for amino acid and n is an integer offrom 2 to 40. The amine terminus, shown here as glycine, is attached toa cross-linker CL which terminates in an antibody-binding group ABG. Thelatter can be any protein binding group such as a maleimide,haloacetamide, isothiocyanate, succinimide ester, imidate ester, and thelike. Substituent R in the aminopolycarboxylate is hydrogen or a groupsuch as 4-isothiocyanatobenzyl to which the peptide portion isattachable. The mode of attachment of DTPA, for example, is via an amidebond (as shown in the structure above) by reaction of the ε-amine ofD-lysine with DTPA dianhydride, or via an isothiourea bonding toisothiocyanatobenzyl DTPA (with the peptide attached to R). It is knownfrom the work of Franano et al., Nucl. Med. Biol. 21: 1023-34 (1994) andothers that the amide bond or isothiourea bond between DTPA and ε-amineof lysine is inert (nonmetabolizable) in the lysosomes. It is also knownthat antibodies radiometalated by, for example, indium or yttrium via anaminopolycarboxylate such as DTPA as a metal chelator are residualized.This phenomenon, documented by Stein et al. (see above) and others, isdue to the hydrophilic nature of the metal chelate as well as its chargeand molecular weight, which all contribute to residualization in alysosomal compartment. This invention uses amino-polycarboxylate on aniodinatable and nonmetabolizable peptide template as one method ofproducing residualizing iodine label. To this end, D-lysine, which iscoupled to an aminopolycarboxylate, is elaborated on the amino end byattaching a D-tyrosine, thus producing a totally inert adduct, whichwhen iodinated and attached to antibodies via a cross-linker at theamine terminus of the said peptide, results in a residualizingradioiodine. When radioiodinated and coupled to a lymphoma antibody LL2the product resembles the same antibody labeled with indium-111 in termsof retention to a lymphoma cell line in vitro, and an enhanced retentioncompared to the same antibody which is conventionally radioiodinated(see Example-5).

Peptides of this category can be readily synthesized on a solid support,either manually or using an automated peptide synthesizer. The number ofamino acid units can be 2-40, with the provision that the DTPA anchoringamino acid is D-lysine or D-arginine or D-ornithine, and that this aminoacid is directly attached to a D-tyrosine. When the peptide containsmultiple D-tyrosines (which is useful for enhancing specificactivities), each tyrosine is attached to D-amino acids. The amineterminus of the peptide can be glycine or an L or D amino acid, and isattached to a cross-linker for coupling to antibodies.

In the reaction sequence shown in SCHEME III, a substitutedaminopolycarboxylate such as DTPA is used to couple radioiodine toantibody. A backbone-substituted DTPA such as 4-nitrobenzyl DTPA(structure on left in SCHEME III where n=0,1,2) is a logical startingpoint for the synthesis. A dianhydride is prepared from nitrobenzylDTPA, and is opened with D-tyrosine under basic non-aqueous conditionsin DMSO or DMF. The nitrobenzyl group in this substrate easily isconverted to an isothiocyanatobenzyl group in a 2-step process ofcatalytic hydrogenation and reaction with thiophosgene (productstructure not shown). This substrate is first radioiodinated and thencoupled to antibody between pH 8-9. Although exemplified by abifunctional DTPA as the starting material, the method is applicable tothe use of bifunctional EDTA or bifunctional TTHA as a startingmaterial.

In another approach, a tyramine and an antibody-binding moiety form partof the aminopolycarboxylate structural unit. This is illustrated by thebifunctional structuresN,N-bis(carboxymethyl)-N′[2-(p-hydroxy-phenyl)ethyl]-2-[p-isothiocyanatobenzyl]ethylenediaminesA and B of the reaction scheme shown. Briefly, the synthesis involveselaborating 4-nitrophenylalanine by first reducing the carboxyl group toalcohol by, for example, using borane as a reducing agent, followed bydialkylation, and oxidation of the alcohol group to an aldehyde viaSloan oxidation with oxalyl chloride in DMSO followed by reductivecoupling to tyramine, and finally converting the nitrobenzyl group to anisothiocyanatobenzyl group. These residues are first radioiodinated andthen coupled to antibody lysine groups or to thiolated antibodies. Thepresence of the basic amino groups and the carboxylic acid groups in theprepared conjugate aids residualization within the acidic lysosomeenvironment.

In the reaction sequence shown in SCHEME V, one or more thiol groups areintroduced into an antibody such as a monoclonal antibody (Mab) by oneof two illustrative methods. In the first, a disulfide bond reducingagent, such as dithiothreitol (DTT), effects either partial or completecleavage of heavy chain disulfide bonds. Alternatively, one or morethiol groups

are introduced with a linker. A protected tertiary thiol is preferred,such as a succinimidyl 2-[N-(3′-methyl-3′-thioacetyl)butamidyl]acetate(1), the thioester of which is then cleaved with hydroxylamine asdescribed by Govindan et al. in Bioconjugate Chemistry, 7:290-297, 1996.

The resultant thiolated antibody is linked to a hydrazide using amaleimide/hydrazide conjugate, e.g.,4-[4′-(N-maleimidyl)-phenyl]butyrylhydrazide (MPBH, 2) or4-(N-maleimidylmethyl)cyclohexane-1-carboxyhydrazide (M₂C₂H, 3).

Finally, the hydrazide is coupled to a radioiodinated, carbohydrate,e.g., dimelibiitol tyramine (DMT, 4) that has been oxidized, for exampleby enzymatic reaction with galactose oxidase.

This oxidized carbohydrate optionally is further stabilized by areductive amination reaction to form radioiodinated DMT-Mab-conjugate.

One advantage of the present invention is that, in contrast to Schiffbase adducts of oxidized carbohydrates formed from simple primary aminessuch as those from lysine, the hydrazone conjugates according to thepresent invention do not require reduction of the imine (hydrazone)function for stabilization. Hydrazones are resistant to hydrolysis underphysiological conditions, while Schiff bases are much more easilyhydrolyzed.

In the reaction sequence shown in SCHEME VI, an oxidized carbohydrate,e.g., DMT, couples with a maleimide/hydrazide conjugate, e.g., MPBH (2)or M₂C₂H (3) to form a maleimide dimelibiitoltyramine. A maleimide groupis alternatively introduced by first reductively coupling2-aminoethylcarbamate to oxidized dimelibiitoltyramine or oxidizedmelibiitoltyramine, followed by deprotection of the remaining primaryamino group and its further conversion to maleimide. The tyramineresidue is iodinated either before or after this reaction. One or morethiol groups are introduced to an antibody by one of two methods asdescribed above. Finally, the maleimide is coupled to the antibody toform a stable radioiodine DMT-antibody conjugate.

Oxidation with a periodate such as sodium or potassium periodate cancreate aldehyde functionalities on the carbohydrate, although compoundscontaining two or more keto or hydroxyl groups attached to adjacentcarbon atoms tend to cleave between these two carbons. Periodateoxidation can cause extensive isomerization and even decomposition of acarbohydrate chain. For these reasons it is preferred to oxidizecarbohydrate with an enzyme such as galactose oxidase, which canintroduce one aldehyde functionality into the carbohydrate withoutcausing other structural changes.

The reaction sequence of SCHEME VII shows coupling of radiolabelledtyramine carbohydrate, such as DMT (4), or MT with a substitutedcyanuric chloride (CC) via displacement of a chlorine on the cyanuricdichloride. The first chlorine of cyanuric chloride is very reactive,the second chlorine of monosubstituted CC is somewhat less reactive,while the remaining chlorine in the disubstituted CC is relativelyunreactive. According to one advantageous embodiment, a monosubstitutedCC is prepared under non-aqueous conditions, from equimolar quantitiesof CC and a maleimide-containing amine, e.g. monosubstituted CC analog(CC analog 5, below).

From the monosubstituted CC depicted above, only one chlorine is readilyavailable for reaction with a radioiodinated carbohydrate such asI-125-DMT. Furthermore, the maleimide group, being thiol-reactive, cancouple subsequently to a thiolated antibody. The high reactivity ofthiol toward maleimide obviates low yield and aggregation problems thatresult from protracted reaction of antibody amine with DMT-derivatizedCC.

Although an Iodine-125 (I-125) radioisotope exemplifies the embodimentsshown in SCHEME I-VII, the methods of the present invention areapplicable to any iodine isotope. I-123 is especially preferred for usein tumor imaging, Iodine-131 (I-131) is especially preferred for use intumor therapy and I-125 is preferred for short-range detection of tumormargins, e.g. for intraoperative, intravascular or endoscopicprocedures.

The incorporation of radioiodine into a peptide or carbohydrate of thepresent invention is carried out by an electrophilic substitutionreaction. This reaction is fast and allows coupling in dilute solutionsof radioiodine. The reaction generally requires oxidation of iodine ionsto produce an electrophilic radioiodination reagent. Methods foroxidizing halide ions are well known in the art and are described byWilbur (see above). Although the invention exemplifies sodiumiodiode/iodogen combinations, the method is not limited to this reagentcombination.

Reactions of substituted cyanuric dichloride are preferably carried outat neutral pH. Neutral pH is defined as a pH between pH 4.5 and pH 9.5.A neutral pH between pH 6 and pH 8 is especially preferred for theinvention.

Many different kinds of maleimide-hydrazides are known or can be made bythe skilled artisan and are suitable for the present invention.Especially preferred are the two maleimide-hydrazides (2) and (3) shownabove. Other maleimide-hydrazides can be synthesized from a maleimidoN-hydroxy-succinimide ester. Representative maleimido-esters useful forthis purpose are: 3-maleimidobenzoic acid N-hydroxy-succinimide ester,β-maleimidobutyric acid N-hydroxysuccinimide ester, ε-maleimidocaproicacid N-hydroxysuccinimide ester,4-(N-maleimidomethyl)-cyclohexane-1-carboxylic acidN-hydroxy-succinimide ester,4-(N-maleimidomethyl-cyclohexane-1-carboxylic acid3-sulfo-N-hydroxy-succinimide ester and β-maleimidopropionic acidN-hydroxysuccinimide ester.

Any antibody that is specific for a tumor cell surface marker is usefulfor the present invention. This antibody preferably has an affinity fora particular cell type that allows antibody targeting to deliverradioiodine for tumor imaging or for tumor therapy. Particularlypreferred are internalizing pancarcinoma antibodies such as RS7 asdescribed by Stein et al., Cancer Res. 50: 1330-36 (1990), internalizinglymphoma antibodies such as LL2 as described by Pawlak-Byczkowska etal., Cancer Res. 49: 4568-77 (1989) and anti-carcinoembryonic antigenantibodies such as Immu-14 as described by Hansen et al., Cancer 71:3478-85 (1993). All three references are hereby incorporated byreference in their entirety. Also preferred are chimeric, humanized andhuman versions of antibodies and antibody fragments.

The present method is particularly well suited for couplingsulfhydryl-containing monovalent antibody fragments, e.g., Fab-SH orFab′-SH, since they can be generated by reductive cleavage of divalentF(ab)₂ or F(ab′)₂ fragments with an appropriate conventional disulfidereducing agent, e.g., cysteine, dithiothreitol, 2-mercaptoethanol,dithionite and the like. Reduction preferably is effected at pH 5.5-9.5,preferably 6.0-6.8, more preferably 6.1-6.4, e.g., in citrate, acetateor phosphate buffer, and advantageously under an inert gas atmosphere.Reduction is faster at higher pH, but reoxidation is also faster. Anoptimal pH is selected wherein reduction is reasonably rapid, butreoxidation, including the formation of mixed disulfides with thiolreducing agents, is negligible. Care must also be taken to avoid overlypowerful reducing agents that will reduce light/heavy chain disulfidebonds in competition with heavy/heavy chain disulfide bonds withinimmunoglobulin proteins. Cysteine is preferred for such disulfidereductions but other thiols having similar oxidation potentials tocysteine also can be used. The ratio of disulfide reducing agent toprotein is a function of interchain disulfide bond stabilities and mustbe optimized for each individual case. Cleavage of F(ab′)₂ antibodyfragments is advantageously effected with 10-20 mM cysteine and aprotein concentration of about 10 mg/ml.

Cleavage of divalent antibody fragments can be monitored by, forexample, size exclusion HPLC, to adjust conditions so that Fab or Fab′fragments are produced to an optimum extent, while minimizinglight-heavy chain cleavage. Eluate from a sizing gel column can be useddirectly or, alternatively, the Fab-SH or Fab′-SH solution can be keptat low temperature, e.g., in the refrigerator, for several days toseveral weeks, preferably at a pH of 3.5-5.5, more preferably at pH4.5-5.0, and advantageously under an inert gas atmosphere, e.g.,nitrogen or argon.

Optimum reaction conditions of time, temperature, ionic strength, pH andthe like suitable for the coupling reactions of SCHEME 1, 2 and 3 can bedetermined by a minimum of experimentation. In fact, one principaladvantage of the present invention is that the coupling reactions withmaleimide and with substituted cyanuric dichloride can take place easilyat neutral pH. Reaction temperature and ionic strength are likewise notcritical. An important consideration is that non-extreme reactionconditions be chosen which will not denature the specific antibody used.

The invention is described further below by reference to illustrativeexamples.

EXAMPLES Example 1

Preparation of (BOC)Gly-D-Tyr(O-t but)-D-Lys-OH

Fmoc-D-Lysine(Aloc)[0.325 g; 0.72 mmol] is dissolved in 5 ml ofanhydrous dichloromethane (CH₂Cl₂), and mixed with 0.55 ml ofdiisopropylethylamine (DIEA). The solution is then added to 0.5 g of2-chlorotrityl chloride resin in a 20 ml vial and the contents shakenvigorously for 18 h. The reddish slurry is placed in a column assemblyfitted with a frit and a 3-way stopcock which can be used to eitherbubble nitrogen through the slurry for mixing purposes or for drainingsolution off the column and leaving the resin on the column. Thesolution is drained off, and the resin is washed with 3×40 ml ofCH₂Cl₂:MeOH:DIEA (17:2:1), 3×40 ml of CH₂Cl₂, 2×40 ml of DMF, 2×40 ml ofMeOH. The resin is dried under a flow of nitrogen. The Fmoc group iscleaved by adding 40 ml of 5% piperidine in 1:1 (v/v) CH₂Cl₂-DMF for 10minutes, draining the solution off, and continuing cleavage with 20%piperidine in CH₂Cl₂-DMF for 15 minutes. This is followed by a washcycle with 40 ml DMF, 40 ml isopropanol(IPA), 40 ml NMP(N-methylpyrrolidone), 40 ml IPA and 4×40 ml NMP. The resin is thenreacted with 1.8 mmol of activated Fmoc-D-tyrosine (O-t but) for 40minutes. The activation is carried out using 0.827 g (1.8 mmol) ofFmoc-D-tyr(O-t but), 0.269 g of HOBT in 4 ml of NMP, adding to the clearsolution 0.31 ml of diisopropylcarbodiimide (DIC), and keeping atambient temperature for 20 minutes. After this period, 3.6 mmol (0.62ml) of DIEA is added, and the reaction is continued for 25 minutes. Thewash sequence, following Fmoc cleavage and subsequent wash sequence, areas described above. A second coupling using activated BOC-glycine(derived from 0.376 g or 3 mmol of Boc-glycine) is carried out in ananalogous manner. The Aloc group is removed using a solution of 0.1547 gof tetrakis(triphenylphosphine)palladium(0) in a mixture of CH₂Cl₂ (40ml): AcOH (2 ml) and DIEA (5 ml), followed by the addition of 5 ml oftributyltinhydride. After the usual wash sequence, the peptide iscleaved from the resin with 10 ml of acetic acid-trifluoroethanol-CH₂Cl₂(1:1:8 v/v). The cleaved peptide solution is concentrated to 0.25 g ofthe title compound (gummy product). The product exhibits a single peakwith a retention time of 7.10 min. on analytical reverse phase HPLCElectrospray mass spectrum shows the M+H peak at m/e 523 (positive ionmode) and the M−H at m/e 521 (negative ion mode).

Example 2

Preparation of Gly-D-Tyr-D-Lys(ITC-Bz-DTPA)-OH

0.053 g (0.1 mmol) of the product from step-1 is mixed with ITC-Bz-DTPA(81 mg of 80% DTPA content; 20% excess)in water-dioxane, and the pH isadjusted to 8.5. The solution is incubated for 2.5 h at 37° C. (bath).More ITC-DTPA (41 mg) is added, and the pH is readjusted to 8.56. Thesolution is then incubated for 2 h at the same temperature. PreparativeHPLC purification on reverse phase column using a gradient elution ofwater (0.1% TFA)/90% acetonitrile-water (0.1% TFA) furnishes 30 mg of(BOC)Gly-D-Tyr(O-tbut)-D-Lys (ITC-Bz-DTPA)-OH as a colorless solid.Analytical reverse phase HPLC shows a single peak with a retention timeof 7.54 min. Mass spectrum analysis revealed a M+H peak at m/e 1063(positive ion mode) and the M−H peak at m/e 1061 (negative ion mode).This material is then treated with a mixture of TFA/CH₂Cl2/anisole for 1h, and the BOC- and Tyr(O-t but) protecting groups are cleaved off. Thetitle compound is precipitated by adding the reaction mixture to ethylether. The HPLC retention time was 5.31 min. Mass spectrum analysisshowed AN M+H peak at 907, and AN M−H at 906.

Example 3

Preparation of (MCC)Gly-D-Tyr-D-Lys(ITC-Bz-DTPA)-OH

0.025 g (0.0138 mmol) of the product from Example-2 is dissolved in 0.5ml of 0.1 M sodium phosphate pH 7.0. To this, 0.03 g of commerciallyavailable sulfosuccinimidyl 4-(N-maleimidomethyl)-1-carboxylate(sulfo-SMCC) is added and the pH is-raised to 7.17. The clear solutionis stirred for 1 h. Preparative HPLC on a preparative reverse phasecolumn using the same gradient elution as in Example-1 yields 0.0054 gof the title compound [where MCC stands for the4-N-maleimidomethyl)-1-carbonyl moiety]. The retention time of thepurified material (analytical RP column) is 6.36 minutes. Electrospraymass spectrum analysis showed aN M+H peak at m/e 1126 and an M−H peak atm/e 1124.

Example 4

Radioiodination of Product from Example 3, Conjugations to a DTT-reducedMonoclonal Antibody IgG [LL2], and to a DTT-reduced Monoclonal AntibodyIgG [RS7]

10 nanomoles of product of Example-3 is radioiodinated with 1.72 mCi ofI-125-sodium iodide by an iodogen iodination method. The labeledsubstrate is transferred to a second vial, and treated with 60 nmol of4-hydroxyphenylacetic acid, followed by reaction with 0.6 mg of ananti-lymphoma antibody [LL2] previously reduced with dithiothreitol togenerate thiol groups by reduction of one or more interchain disulfidebonds of the antibody. After 1-2 h of reaction, the solution is made 5mM in sodium tetrathionate, incubated for five minutes, and purified ona centrifuged size-exclusion column of Sephadex™ 50/80 in 0.1 M sodiumphosphate pH 7. Based on the amount of activity placed on the column, a37.4% recovery of radioactivity of antibody-bound material is obtainedwhich was 95% pure as determined by size-exclusion chromatography viaHPLC. The specific activity achieved in this procedure is 0.94 mCi/mg.

In a variation of this process, 10 nmol of the product of Example-3 isradioiodinated with 2.24 mCi of I-125 sodium iodide using chloramine Tas oxidant for 1-2 minutes. Unused active iodine is quenched with4-hydroxyphenylacetic acid, diluted with potassium iodide and reactedwith 0.5 mg of DTT-reduced LL2 for 15-40 minutes. The work up andchromatography is as described above. This yields 41%-43% overall yieldwith a final specific activity in the 1.98-2.09 mCi/mg range.

The product of Example-3 (10 nmol) is radioiodinated with 1.46 mCi ofNal (I-125) using iodogen as oxidant, and the radioiodinated material isconjugated to DTT-reduced RS7 (0.5 mg). An overall yield (afterpurification) of 29.3% at a final specific activity of 1.0 mCi/mg isobtained. Analysis of the purified material on analytical SEC HPLCshows >98% of radioactivity associated with the antibody.

Example 5

In vitro Binding Studies

The product of Example-4 is incubated with Raji cells (10⁶ cells/ml inDulbecco's double eagle medium) in a sterile incubator maintained at 37°C. After 2 h, the cells are pelleted by centrifugation, and thesupernatant solution is discarded. The cells are washed three times withcold media. The washed cells are resuspended in fresh media and placedin an incubator. At various time points, a known volume of the cellsuspension is removed, pelleted and the activity associated with thecell pellet is determined. The control experiment involves using thesame antibody labeled directly by a chloramine T procedure (negativecontrol) or the same antibody labeled with In-111 (by labeling theproduct of Example-3 with In-111 acetate, followed by coupling toDTT-reduced LL2 as a positive control) The product of Example-4 wasfound to be associated longer with the Raji cells by comparison withdirectly radioiodinated LL2. This retention parallels the retention ofIn-111 on Raji cells. [% initially bound cpm retained: For I-125 labeledLL2: 94.7%(2 h), 63.8% (26 h), 51.1%(48 h)& 35.4%(120 h); for In-111labeled LL2: 89.2%(2 h), 68.1%(26 h), 49.8%(48 h) & 34.1 (120 h).

In a similar fashion, in vitro bindings, to Calu 3 non-small lungadenocarcinoma cell line, of RS7 radioiodinated with residualizing labelof this invention (that is, the product of Example-3 radioiodinated andcoupled to DTT-reduced RS7)and conventionally radioiodinated RS7 werecompared. Data from this Example showed that the residualizing labeexhibited distinctly prolonged retention compared with that of theconventional iodine label.

Example 6 SCHEME IV Preparation ofN,N-bis(carboxymethyl)-N′-[2-(p-hydroxyphenyl)ethyl]-2-[p-isothiocyanatobenzyl]-ethylenediamineA and the corresponding maleimide B

4-Nitrophenylalanine is reduced with borane in THF. Reaction with twoequivalents of tert-butylbromoacetate and anhydrous sodium carbonate inrefluxing acetonitrile furnishes a dialkylated product in 67.9% yieldafter flash chromatographic purification. The 400-MHz proton NMRspectrum of this product is fully consistent with the structuralassignment. The intermediate (0.1 g) is oxidized in high yield toaldehyde using DMSO/oxalyl chloride at −78° C. followed by treatmentwith triethylamine, and the purified product is reacted with tyramine inpresence of sodium cyanoborohydride in aqueous methanol. Thistyramine-appended intermediate (70% overall yield) is characterized byM+H peak at m/e 544 (electrospray mass spectrum, positive ion mode).Catalytic hydrogenation of the nitro group to an aniline derivative(product is characterized by mass spectrum), followed by a 2-stepreaction sequence (involving deprotection of carboxyl protecting groupsusing hydrochloric acid, and a subsequent reaction with thiophosgene in3 M hydrochloric acid) gives the isothiocyanate derivative A, which inturn is converted to maleimide derivative B in two steps (reaction withethylenediamine, followed by treatment with SMCC as described inExample-3).

Example 7

Radioiodinations of Lymphoma Antibody (LL2) using I-125-labeled A orI-125-labeled B (A & B of SCHEME4)

Radioiodination of 10 nmol of A (Na¹²⁵I/iodogen) followed by quenchingof unreacted radioiodine with 40 nmol of aqueous phenol, and subsequentreaction with 1.37 mg of LL2 at pH 8-8.2 for 3 h at 37° C. gives anincorporation of 32.8%. Experiments are carried out using lesser amountsof the antibody to increase the specific activity. An incorporation of23.7% at a specific activity of 1.1 mCi/mg, and 24.1% incorporation at aspecific activity of 1.4 mCi/mg are obtained. The aggregate content wasas low as 2%. Using B, and reduced LL2 (reduction carried out as inExample-4), an incorporation of 28.4% at a specific activity of 0.95mCi/mg is obtained, with negligible aggregation.

Example 8

Preparation of Dimelibiitoltyramine

The title product is prepared using 2.23 g (6.55 mmol) of melibiose,0.089 g (0.657 mmol) of tyramine and 0.169 g (2.63 mmol) of sodiumcyanoborohydride in 5 ml of borate buffer pH 9 at 65° C. for 18 h. Thesolution is acidified to pH 4.6, and purified on a 2.5 cm (o.d.) and 10cm height column of Dowex 50-X2 cation exchange resin packed in 0.05 Mammonium acetate pH 4.6. Elution is with the same buffer, followed by alinear gradient of 1 L of water and 1 L of 1 M ammonium hydroxide at aflow rate of 2-3 ml/minute. Fractions of 4 ml each are collected. Assayof fractions by UV absorbance at 280 nm gave the elution profile. Theelution profile contains a single peak. Accordingly, eluate fractions 60through 70 are pooled, evaporated and lyophilized to obtain 0.98 g of acolorless solid comprising of the title product and an inorganic salt.The content of dimelibiitoltyrmine in an aqueous solution is determinedusing the absorbance value at 280 nm. The electrospray mass spectrum ofthe lyophilized material shows the correct M+H peak at m/e 790.

Example 9

Radioiodination of LL2 Using I-125-DMT

In one experiment, 10 nmol of DMT is labeled with I-125 (iodogenmethod). The iodinated DMT is then oxidized with about 10 units ofgalactose oxidase at 30° C. for 2.5 h. The oxidized ¹²⁵I-DMT is reactedwith equimolar (10 nmol) of LL2 and 20 mM sodium cyanoborohydride at thesame temperature for 18 h. Incorporation is 18.7%, which is reproducible(20.7% in a second run).

Example 10

In vitro Binding of Product of Example 9 to Raji Cells

The experiment is carried out analogously to that described inExample-5, using directly radioiodinated antibody (by a chloramine-Tprocedure) as a control. The results revealed significant retention, onRaji cells, of the product of Example-9 on Raji cells compared to thatof the control over a 170 time period.

Example 11 SCHEME I

Reaction of Thiolated Antibody with a Maleimide-containing Hydrazide andthen with Oxidized Carbohydrate

In the first step of this scheme, IgG disulfide bond(s) are reduced withdithiothreitol. Briefly, 0.55 ml of an internalizing anti-lymphomaantibody LL2 described by Pawlak-Byczkowska, et al., Cancer Research 49:4568-77 (1989) is mixed with an equal volume of sodium phosphate bufferat pH 7.4, 0.11 ml of 0.5 M borate buffer pH 8.5 and 6 ul of 0.4 g/mldithiothreitol in water. The reaction mixture is mixed vortex andincubated at room temperature for 30 minutes. The thiol reduced antibodyis then purified by size-exclusion chromatography by passing the reducedprotein solution through a Sephadex™ 50/80 resin equilibrated inphosphate buffer at pH 7.0.

In the second step of this scheme, a hydrazide group is introduced intothe LL2. Ten equivalents of hydrazide-maleimide M₂C₂H dissolved indimethyl formamide are added to the prepared antibody solution for eachSH group on the IgG. Incubation is continued at 37° C. for two hours. Toquench the reaction, a 50-fold molar excess of N-ethylmaleimide is addedand incubation is continued for another 30 minutes at 37° C. The treatedIgG is then purified by size-exclusion chromatography.

In the third step of this scheme, IgG is conjugated withdimelibiitol-^(125/131)I-tyramine. Dimelibiitol tyramine is firstradioiodinated with radioactive sodium iodide using iodogen. Theiodinated dimelibiitol tyramine is oxidized with galactose oxidase byone of the procedures summarized by Strobel et al., Arch. Biochem.Biophys. 240: 635-45 (1985). The treated IgG is conjugated to thealdehyde group of oxidized dimelibiitol tyramine at a 1:1 molar ratio inphosphate buffer at pH 7.7. After two hours, sodium cyanoborohydride isadded to a final concentration of 20 mM and the reaction mixture isincubated for an additional one hour. The conjugate that containsdimelibiitol-^(125/131)I-tyramine is again purified by size-exclusionchromatography at pH 7.4.

Example 12 SCHEME I

Thiolation of Antibody and Subsequent Coupling Via aMaleimide-containing Hydrazide

In the first step of this scheme, at least one thiol group is introducedinto an antibody by reaction with a thiolating reagent (1). Thethiolating reagent is dissolved in dimethylformamide to a final dimethylformamide concentration of 5% vol/vol. The thiol content of the preparedantibody is determined by Ellman's assay. The thiolated antibody isequilibrated in phosphate buffer at pH 7.5 and then allowed to reactwith a four- to ten-fold excess of the maleimide-hydrazide reagentM₂C₂H. After reaction between antibody and reagent MPBH, the conjugateis purified by size exclusion chromatography. The hydrazide-introducedantibody is then coupled to oxidized dimelibiitol-¹²³I-tyramine byfollowing the procedures outlined in steps two and three of Example 11above.

Example 13 SCHEME II

Introduction of a Maleimide-containing Hydrazide into OxidizedCarbohydrate Followed by Coupling to Antibody

In this scheme oxidized dilactitol-¹²³I-tyramine is incubated with atwo-fold excess of M₂C₂H in phosphate buffer between pH 6 and pH 7 forone hour. Then the thiolated antibody prepared as described above inExample 1 or Example 2 is added to the incubation mixture. After anadditional 30 minutes of incubation at ambient temperature,iodoacetamide is added and incubated for another 30 minutes to quenchunreacted thiol. Sodium cyanoborohydride is added to a finalconcentration of 10 mM and allowed to incubate for one hour. Preparedantibody is purified from the final reaction mixture by size exclusionchromatography.

Example 14 SCHEME III

Cyanuric Dichloride Coupling of Antibody with Residualizing Label

In this scheme a substituted cyanuric dichloride is used to coupleantibody to residualizing label. The cyanuric dichloride derivative isdissolved in dimethyl formamide or in dimethyl sulfoxide and then addedto a water solution of dilactitol-¹²³I-tyramine.

In the case of coupling with a cyanuric dichloride analog, the cyanuricdichloride analog is first incubated with the residualizing label for 90minutes at 37° C. After cooling, antibody solution in a phosphate bufferis added and the mixture is incubated for three hours at 37° C. The pHof the antibody buffer solution is adjusted so that the final reactionpH is between pH 7-8 when coupling to antibody. The final pH is betweenpH 6-7 for the case of coupling with thiolated antibody. The antibody ispurified from other reactants by size exclusion chromatography.

For each of these examples, the conjugation efficiency is determined bymeasuring the amount of I-123 incorporated into protein and the amountof protein recovered. The degree of IgG aggregation is determined bymolecular size analysis of prepared IgG using analytical size exclusionHPLC or polyacrylamide gel electrophoresis in the presence of sodiumdodecyl sulfate.

The optimum amounts of residualizing agent, antibody and other reagentsin these reactions are determined by varying their molar ratios andmeasuring the specific activities, binding affinities and amounts ofconjugate formed under each reaction condition.

Example 15

Comparative Biodistributions of Lymphoma Antibody LL2, Labeled withResidualizing I-125 Label Derived from Product of Example-3 or withI-131 Using Conventional Chloramine T Method, in Nude Mice Bearing RamosHuman Tumor Xenografts

Tumors were grown in 4-week old female nude mice using the Ramos tumorcell line. After two weeks tumor reach the size of about 0.1-0.2 gram.At this stage, groups of five mice were administered about 10 μCi eachof the two iodine labels (I-125 residualizing & I-131 conventional),both contained in the same vial. The dual label gives the more precisecomparisons since variations of antibody dose and tumor size do notexist. Animals are sacrificed at 1 day, 3 days, 5 days, 7 days and 10days post-administration of the labeled antibody. Various organsincluding tumor are excised; the radioactivity associated with theorgans are expressed as a percentage of injected dose per gram (% ID/g).Data obtained from this Example show prolonged retention and superiortumor:non-tumor ratios of accretion for residualizing I-125 label versusconventionally prepared (CT method) I-131 label.

Example 16

Preparation of the Radioiodinated Carbohydrate-appended Peptide: LL2-CCGly-D-Tyr-D-Lys-Mel

A functional group-protected peptide BOC-Gly-D-Tyr(O-tBu)-D-Lys is firstprepared by solid state peptide synthesis using a conventional Fmocstrategy. The product is reductively coupled, at the ε-amino group ofthe lysine, using a ten-fold molar excess of melibiose and sodiumcyanoborohydride in an aqueous buffer at a pH of 8-9. The BOC and thet-butyl protecting groups of the resultant purified product are removedusing trifluroacetic acid and free-radical scavengers, and the productthus obtained is coupled to the cross linker sulfo-SMCC. This finalproduct is radioiodinated and conjugated to Mab LL2 reduce under mildconditions to cleave an average of one disulfide bond to sulfhydrylgroups. The carbohydrate-appended peptide illustrated here is abifunctional variation of dimelibiitoltyrosine. This method also allowspreparation of bifunctional variations of melibiitoltyrosine as well asmono and di-adducts of other reducing sugars, for example, lactose, byvarying the molar excess and/or the specific reducing sugar used.

From the foregoing descriptions, one skilled in the art can easilyascertain the essential characteristics of this invention and, withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions.

What is claimed is:
 1. A carbohydrate-appended peptide useful forradioiodinating an antibody, comprising: (a) a peptide that comprises atleast one D-tyrosine, an amino terminus, a carboxy terminus formed froma D-lysine and no contiguous L-amino acids between the D-tyrosine andthe carboxy terminus; (b) a reducing carbohydrate conjugated to thepeptide via an ε-amino group of the D-lysine to form acarbohydrate-appended peptide; and (c) a linker group for covalentlybinding said carbohydrate-appended peptide to an antibody.
 2. Thecarbohydrate-appended peptide of claim 1, further comprising aradioiodine atom covalently bound to the D-tyrosine residue.
 3. Thecarbohydrate-appended peptide of claim 1, wherein said linker group iscapable of reacting with a sulfhydryl residue of an antibody to form acovalent bond.
 4. The carbohydrate-appended peptide of claim 1, whereinsaid peptide contains 2-40 amino acids.
 5. The radioiodinatedcarbohydrate-appended peptide of claim 2, wherein said peptide contains2-40 amino acids.
 6. The carbohydrate-appended peptide of claim 1,wherein said D-tyrosine is directly linked to said D-lysine.
 7. Thecarbohydrate-appended peptide of claim 1, wherein said carbohydrate isselected from the group consisting of melibiose and lactose.
 8. Theradioiodinated carbohydrate-appended peptide of claim 2, wherein saidcarbohydrate is selected from the group consisting of melibiose andlactose.
 9. The carbohydrate-appended peptide of claim 1, wherein saidcarbohydrate is melibiose.
 10. The radioiodinated carbohydrate-appendedpeptide of claim 2, wherein said carbohydrate is melibiose.
 11. Thecarbohydrate-appended peptide of claim 1, wherein said linker group is amaleimide, haloacetamide, isothiocyanate, succinimide ester, cyanuricchloride, or imidate ester.
 12. An antibody conjugate comprising thecarbohydrate-appended peptide of claim 1 covalently bound to an antibodythrough said linker.
 13. An antibody conjugate of claim 12, furthercomprising a radioiodine atom covalently bound to a D-tyrosine residueof said carbohydrate-appended peptide.
 14. A carbohydrate-appendedpeptide useful for radioiodinating an antibody, comprising: (a) apeptide that comprises at least one D-tyrosine, an amino terminus, acarboxy terminus, and no contiguous L-amino acids between the D-tyrosineand the carboxy terminus and wherein the carboxy terminus is a D-lysine,D-arginine or D-orthnithine; (b) a reducing carbohydrate conjugated tothe peptide via the ε-amino group of the D-lysine or the side chain ofD-arginine or D-ornithine to form a carbohydrate-appended peptide; and(c) a linker group for covalently binding said carbohydrate-appendedpeptide to an antibody.